SLITRK1-mediated noradrenergic projection suppression in the neonatal prefrontal cortex

SLITRK1 is an obsessive-compulsive disorder spectrum-disorders-associated gene that encodes a neuronal transmembrane protein. Here we show that SLITRK1 suppresses noradrenergic projections in the neonatal prefrontal cortex, and SLITRK1 functions are impaired by SLITRK1 mutations in patients with schizophrenia (S330A, a revertant of Homo sapiens-specific residue) and bipolar disorder (A444S). Slitrk1-KO newborns exhibit abnormal vocalizations, and their prefrontal cortices show excessive noradrenergic neurites and reduced Semaphorin3A expression, which suppresses noradrenergic neurite outgrowth in vitro. Slitrk1 can bind Dynamin1 and L1 family proteins (Neurofascin and L1CAM), as well as suppress Semaphorin3A-induced endocytosis. Neurofascin-binding kinetics is altered in S330A and A444S mutations. Consistent with the increased obsessive-compulsive disorder prevalence in males in childhood, the prefrontal cortex of male Slitrk1-KO newborns show increased noradrenaline levels, and serotonergic varicosity size. This study further elucidates the role of noradrenaline in controlling the development of the obsessive-compulsive disorder-related neural circuit.

in a Sema3a-induced endocytosis NRP/L1CAM complex. Based on this model, S330A would be predicted to be less efficient than WT Slitrk1 in suppressing endocytosis, but this is not observed. This apparent discrepancy should be discussed on page 19.
Reviewer #3 (Remarks to the Author): This manuscript is an extension of authors' previous study on SLITRK1 knockout (KO) mice, which described the altered anxiety-like behavior and abnormalities in noradrenergic functions. They employed both gain-of-function and loss-of-function studies, revealing that Slitrk1 suppresses the noradrenergic projection connectivities that might be involved in a subset of behavioral deficits. Particularly, they asked whether two SLITRK1 missense mutations linked to schizophrenia/bipolar disorder (S330A and A444S) could disrupt any known SLITRK1 functions (e.g. neurite outgrowth). Moreover, they found that L1-CAM binds to SLITRK1 in a nanomolar affinity. I am impressed by large amount of data from various approaches; but the following points should be completely addressed for consideration in Communication Biology.
Major points: 1. Authors need to present summarized/representative results throughout the manuscript by at least three biological replicates. I noted that there are some experiments where the number of samples is below 3. These should be completely addressed. 2. Some of image qualities are not excellent. For example, in Figure 6D, I don't see that authors could conclude any clear conclusions. More importantly, the control experiments appeared not to work -SLITRK1 was reported to induce VGLUT1 clustering in previous studies. 3. Many representative images do not match with the respective quantification results (e.g. Figure 2A and 2B; Figure 3A and Figure 3B; Figure 6A and 6B). 4. I don't understand why the authors treated the SLITRK1 ECD recombinant proteins into cultured LC neurons in Figure 4. I suppose that authors should employ transfection experiments because SLITRK1 is a transmembrane protein. In addition, the representative images do not clearly reflect the message in the Figure 4B. 5. Figure 8: the authors should present the data that show the direct binding of SLITRK1 with Neurofascin and L1CAM, not just by showing Kd values (panel B). Authors did not present any compelling results to demonstrate the interactions of SLITRK1 with these proteins. More rigorous biochemical and biophysical analyses are critical. 6. I am not persuaded by authors' representative images/data that S330A mutations has phenotypes in many cases. 7. Sexual dimorphism is an important/exciting issue in this field. However, authors did not integrate the major findings related to this issue in the current manuscript. Could authors show for example that differences in the norepinephrine levels between sexes are causally involved in behavioral deficits and neurite outgrowth/synaptic impairments? 8. Data presentations: this is serious -bar graphs should be presented in a consistent manner throughout the paper.
Minor points: 1. Discussion section is too lengthy. Authors need to remove/trim considerable parts and move them to the Introduction section. 2. Authors need to discuss in detail why varicosity size in the NET fibers are opposite in SLITRK1 KO mice during development (i.e., P7 vs. 6M). 3. Page 8, line 4: "but females" should be changed to "but not females" 4. Figure S4: These data should be quantified. 5. Where are the representative images for Figure 3I and 3J in Figure 3H? 6. Where are the representative images for BSA groups in Figure 8F? 7. Statistics: # should be added in the legend of Figure 2B. In addition, † † need to removed. In Figure   2E, † need to corrected. Importantly, authors should be very careful to indicate all the statistics in data and legend (e.g. statistics missed in Figure 2E for MHPG and MHPG/NA groups). 8. Figure 2B: further experiments are necessary for female mice.
Reviewer #4 (Remarks to the Author): Summary: This manuscript reports an array of neurodevelopmental differences in mice lacking SLITRK1, which is a transmembrane protein associated with OCD-related disorders and known to function in neurite outgrowth and synapse formation. The paper builds on the authors' previously published work, which implicated noradrenergic mechanisms in the anxiety-like behaviors of slitrk1-deficient mice. Here, the authors report structural, behavioral, and neurochemical differences in slitrk1-deficient mice, with an emphasis on the neonatal prefrontal cortex. The authors also report two novel missense SLITRK1 mutations associated with schizophrenia and bipolar disorder and link these mutations to structural and functional deficits in the noradrenergic system. The paper is ambitious in scope, technically rigorous, and provides further evidence that cellular and molecular differences in noradrenergic signaling may contribute to numerous neurodevelopmental disorders, including OCD, schizophrenia, and bipolar disorder.

Major points:
This work is of interest to at least three specialized audiences -those who examine SLITRK protein functions in neurodevelopment, those interested in the development of the prefrontal cortex, and those interested in the cellular and molecular changes that contribute to OCD and other pervasive but poorly understood neurodevelopmental disorders.
With 9 Figures and 13 Supplementary Figures, the paper is ambitious in a way that ultimately compromises its cohesion and readability. The authors characterize a knock-out mouse at the behavioral, anatomical, and molecular levels, provide evidence for changes to multiple neurotransmitter pathways, perform a genome wide association study, report novel SLITRK1interacting proteins, and examine the complex molecular mechanisms mediated by these protein interactions. The reason why some figures are relegated to Supplementary files (while others are not) is somewhat difficult to discern. It is an impressive and technically rigorous body of data, but it may benefit from being divided into two smaller, more cohesive manuscripts.
Two general issues regarding the clarity of the manuscript should be addressed. First, the final paragraph of the introduction should be revised and expanded to articulate the overall rationale and major findings of the paper with greater specificity. Second, the Results section could benefit from clear statements of rationale as each experiment is introduced and described. This was done nicely in the Discussion, but bears repeating in the Results as well.
In Figures 2 and 3 (and others), mean values of WT controls are set to 1, but no mention of the absolute values can be found in the figure legends or results text. A supplementary data file containing absolute measurements would be a positive addition to the supplementary materials. Figure 5, which describes the genome wide association study that identified new SLITRK1 missense mutations, may be better suited to the Supplementary data.
The claim that Slitrk1-knockout mice exhibit reduced GABAergic synapse density in the PFC is not adequately supported by the evidence presented in Figure S8. One or more additional inhibitory synapse markers would strengthen this claim considerably.

Minor Points:
In Figures 2-4, the WK labels below the X axes are redundant with the shading of the bars and the associated key. Removing the WK labels would improve the clarity of these figures.
Statistical analyses are appropriate throughout, and the level of experimental detail provided in the Methods section is adequate for reproducibility.

Point-by-point responses to reviewers' comments:
Reviewer #2: Hatayama et al investigate the role of the disease-associated gene Slitrk1 in brain development.
The authors perform an in-depth analysis of behavioral and anatomical parameters in Slitrk1 KO mice. Differences in ultrasonic vocalizations and noradrenergic fiber density in prefrontal cortex (PFC) are observed. Analysis of the molecular mechanisms involved points to interactions of Slitrk1 with L1 family proteins and endocytosis. Overall this is an interesting study that reveals the consequences of impaired Slitrk1 function on the development of noradrenergic circuitry.
Specific comments 1. The manuscript contains a very large amount of data and the results are not always easy to follow. The paper would benefit from reorganization to facilitate reading, for example by reducing supplemental data and grouping data in main figures in line with the text (as an example, now the text moves from Fig. 2 to Fig. 3, then back again to Fig. 2 to discuss metabolite levels and back again to Fig. 3 to discuss VGAT/VGLUT1 staining -this is confusing).
The same applies to the order in which figure panels are discussed (for example, Fig. 4D is discussed before 4C in the text). Some supplemental data is not commented on (for example, Fig.   S2B, vertical screen test significant difference only at P15).
Response: After reading the three reviewers' comments, we realized that the previous manuscript was not well organized and forced the readers to seek the data many times. According to this comment, we have reorganized the results so that the readers can follow the study without unnecessarily going back-and-forth. The Supplementary Figures were limited to only those figures which the readers could follow the study without seeing, and the number was reduced from 12 to 9.
References to supplemental data sometimes appear incomplete (for example, page 7, reference to Supp. FigS4 for increased density of NET-positive fibers likely should include Fig. S5 as well).
Finally, the text should be proofread to check for omissions, typos, and to make sure sentences flow well (as an example, 'but females' page 8 probably means 'but less in females' or similar).
Response: We have corrected and checked the reference to the supplemental data. Several instances, including the one pointed out, were corrected to improve the flow. The revised text was finally corrected by a professional scientific Editor once again. Fig. 2A and S5A are the same for P7 WT and KO panels. From the text it appears that there should be data on P3 but this is missing.

Images in
Response: Figure  Response: Statistical tests were performed by two-tailed unpaired t-test and two-way ANOVA (genotype and sex as main factors). A data point indicates a mean value for each mouse. The mean values were derived from three qPCR experiments that were performed in duplicate. In this revision, we carried out multiple test correction as per the Benjamini−Hochberg procedure and confirmed that the changes in the three genes were significant after the multiple test correction. 4. Page 10, 'Compared with control treatment, Sema3A treatment led to decreased and increased complexity of proximal and distal neurites, respectively (Fig. 4C); moreover, it decreased the neurite length (Fig. 4D). However, there was no significant between genotype difference in the complexity ( Supplementary Fig. S10-Sema3a).' It is unclear to me how the data in 4C, D are related to S10 and what is meant by this sentence.
Response: We apologize for the confusion caused by our unclear description. "Control treatment" indicated to the BSA-treated one, referring to the BSA-Sema3A difference in the KO samples.
To avoid confusion, we have moved the graphs for between-genotype difference (previous Figure   S10) to the bottom of new Figure 6c and referred to the individual graph in the main text. 5. Fig. 4C, D: Slitrk1-ECD treatment has no effect on intersections and branch length in Slitrk1 KO neurons -please discuss.
Response: We agree that this is an important finding. In the last paragraph of Molecular mechanism underlying Slitrk1-mediated suppression of NA projections chapter, we have discussed this as follows: "The fact that the neurite suppressive effect of SLITRK1 ECD requires Slitrk1 in LC neuron suggests that Slitrk1 and Slitrk1 ECD may compete for the L1 family proteins. However, we cannot exclude other possibilities, such as homophilic interaction via Slitrk1 ECDs or it's binding to unidentified targets." 6. Fig. 6E: the effect of A444S is not significant, yet it is concluded that both S330A and A444S impair inhibitory synapse-inducing ability of Slitrk1, this is also repeated in Discussion. This should be corrected.
Response: This comment may have been raised due to an unclear indication of statistical significance in Figure 6E graph. In the bar graphs of the revised manuscript, we have consistently used rotated-square-brackets to indicate the two groups for the comparison. The corresponding The differences between WT, S330A and A444S are very small, how was significance determined? From the legend it appears that a t-test was used, which is incorrect for multiple conditions here, further it is not clear if data points represent individual cells or independent experiments, in case of the former, corrections should be made to account for independent experiments. In general, statistical analysis should be carefully checked throughout the paper.
Response: In the revised manuscript, we carried out Dunnett's or Steel's test for the mutant analysis (one-to-many comparison). This has also been written in Experimental design and statistical analysis subsection of the Methods section and in the Figure legend. 7. Fig. 7: this experiment lacks a proper control such as GFP or a non-relevant membrane protein.
Currently data is compared to a frame-shifted Slitrk1 mutant that is predicted to generate a cterminally truncated ECD fragment. A proper control to account for the experimental procedure  Fig. 8F, H: the authors show Slitrk1 binding affinity for L1 family member Neurofascin is reduced for Slitrk1 S330A (Fig. 8B). According to the model the authors discuss, Slitrk1 might displace L1CAM in a Sema3a-induced endocytosis NRP/L1CAM complex. Based on this model, S330A would be predicted to be less efficient than WT Slitrk1 in suppressing endocytosis, but this is not observed. This apparent discrepancy should be discussed on page 19.
Response: Because the binding to L1 was comparable between SLITRK1 WT and S330A as shown above, the endocytosis-suppressing activities of the two proteins may be equivalent.

Reviewer #3:
This manuscript is an extension of authors' previous study on SLITRK1 knockout (KO) mice, which described the altered anxiety-like behavior and abnormalities in noradrenergic functions.
They employed both gain-of-function and loss-of-function studies, revealing that Slitrk1 suppresses the noradrenergic projection connectivities that might be involved in a subset of behavioral deficits. Particularly, they asked whether two SLITRK1 missense mutations linked to schizophrenia/bipolar disorder (S330A and A444S) could disrupt any known SLITRK1 functions (e.g. neurite outgrowth). Moreover, they found that L1-CAM binds to SLITRK1 in a nanomolar affinity. I am impressed by large amount of data from various approaches; but the following points should be completely addressed for consideration in Communication Biology.
Major points: 1. Authors need to present summarized/representative results throughout the manuscript by at least three biological replicates. I noted that there are some experiments where the number of samples is below 3. These should be completely addressed.
Response: We noticed that the previous Supplementary Figure 8 contained the results of n = 2 and n =3. The figure and related description were removed from this study. In relation to this revision, we removed the quantitative analyses of synapses in Slitrk1 KO mice (previous Figure   3H-J, S9D, E). This was done because we had to simplify the entire study in response to the comments from the other reviewers. The synaptic phenotype will be described elsewhere.
2. Some of image qualities are not excellent. For example, in Figure 6D, I don't see that authors could conclude any clear conclusions. More importantly, the control experiments appeared not to work -SLITRK1 was reported to induce VGLUT1 clustering in previous studies.
Response: We replaced Figure 6D (new Supplementary Figure 7) images. In our experiments, SLITRK2 induced both inhibitory and excitatory synapses (new Supplementary Figure 7). In this regard, a control may be working. Concerning the discrepancy with previous reports, several experimental parameters are different among the studies (see the Table below). Most critically, previous studies used artificial signal peptide sequence and N-terminal epitope tag, and the timing of the co-culture was earlier than ours. The difference between SLITRK1 and SLITRK2 in our condition may reflect differences in some inherent properties of these proteins. We feel that it would be worth reporting the result in this condition.
3. Many representative images do not match with the respective quantification results (e.g. Figure   2A and 2B; Figure 3A and Figure 3B; Figure 6A and 6B).
Response: We agree with this comment. We have replaced the images in Figure 2A  "We also investigated the effect of SLITRK1 ECD because SLITRK1 ECD is known to be cleaved by α/γ secretase at the transmembrane region 17 ". To consider the role of Slitrk1 in LC neurons (cell-autonomous function), we compared the LC neurons from Slitrk1 WT and KO mice.
In the revised figure (new Figure 6b, c), we replaced the representative images in Figure 4B and merged graphs in Figure 4C and Figure S10. We hope that these corrections can improve the readability and visibility. Response: Accordingly, we performed pull-down assay using purified SLITRK1 ECD, Neurofascin ECD, and L1CAM ECD proteins. The results are indicated in new Supplementary   Figure 8c. The results for the pulldown assay using brain lysate and SLITRK1 ECD are also indicated for L1CAM, Neurofascin, and NCAM (new Supplementary Figure 8b). In addition, we measured the Kd values the SLITRK1 mutants (new Figure 10b), and the original result for mass spectrometry was indicated in new Supplementary Table 2 upon suggestion from another reviewer.
6. I am not persuaded by authors' representative images/data that S330A mutations has phenotypes in many cases.
Response: Representative images for Figure 6D (new Supplementary Figure 7) were replaced with better images. The figures indicating S330A's effect on "later" neurite development (previous Figure S10I-K) were incorporated into new Figure 8 (e-g). These figures most clearly indicate the S330A phenotypes. We agree with the reviewer in that S330A phenotypes are not so drastic. However, this may be a very important mutation with not only pathological, but also evolutionary significance (please see the last paragraph of the Discussion section). We therefore conducted the experiments carefully. In this regard, we added the following description in the Experimental design and statistical analysis subsection of the Methods section: "The experiments for assessing the new SLITRK1 mutations were carried out in a plasmid identity-blinded manner.
The expression constructs for SLITRK1 WT and its mutants were verified by sequencing after each plasmid preparation." 7. Sexual dimorphism is an important/exciting issue in this field. However, authors did not integrate the major findings related to this issue in the current manuscript. Could authors show for example that differences in the norepinephrine levels between sexes are causally involved in behavioral deficits and neurite outgrowth/synaptic impairments?
Response: We agree with the reviewer. We observed several sexual dimorphisms in Slitrk1 KO phenotypes (body weight, USV, NA contents in PFC, and SERT varicosity size in PFC) and discussed the possible basis of this observation (e.g. VMAT2, COMT) in the first subsection of the Discussion. The possible causal relationships were described for the synaptic, behavioral, and developmental phenotypes in the same subsection. In addition, we referred to the male-predominant occurrence of early-onset OCD in the "Excessive NA projections as a disease core mechanism underlying OCRD" subsection.
8. Data presentations: this is serious -bar graphs should be presented in a consistent manner throughout the paper.
Response: Accordingly, we changed the graph design for consistentcy. All data for WT mice are indicated by open circles or bars and those for KO mice are indicated by red circles and bars. Data for SLITRK1 proteins and its mutants are indicated by different colors (WT, blue; S330A, green; A444S, yellow). Statistical significance was indicated in a consistent manner.
Minor points: 1. Discussion section is too lengthy. Authors need to remove/trim considerable parts and move them to the Introduction section.
Response: We moved the background information on the neural circuit basis of OCD and molecular determinants of the monoaminergic fibers from the Discussion to Introduction. This revision may be beneficial for the readers to read the Results more easily.
2. Authors need to discuss in detail why varicosity size in the NET fibers are opposite in SLITRK1 KO mice during development (i.e., P7 vs. 6M).
Response: We added the following discussion in the first chapter of the Discussion section.
"Contrarily, the NET + varicosity size in the PFC of male Slitrk1 KO was larger at P7 but smaller than those of WT at 5W or 6M (Figure 2). While the increase at P7 can be interpreted as a feedback from excessive NA via α2 autoreceptor, the decrease at later stages suggests the presence of some adaptive mechanisms for the excessive NA. As a candidate mediator of such adaptive response, VMAT2 should be noted because VMAT2 is a critical regulator of presynaptic NA storage in brain, and VMAT2 expression is dynamically regulated both during development and upon acute and chronic drug exposure (Eiden and Weihe, 2011)." 3. Page 8, line 4: "but females" should be changed to "but not females" Response: We have corrected it.
Response: We have quantified the NET + fiber area. The results are indicated at the bottom of the images.

5.
Where are the representative images for Figure 3I and 3J in Figure 3H?
Response: We have removed the corresponding figures in this revision. Please see the response to Major point 1.
6. Where are the representative images for BSA groups in Figure 8F?
Response: The representative images for the BSA control experiments were added to Figure 8F (new Figure 10f).
7. Statistics: # should be added in the legend of Figure 2B. In addition, † † need to removed. In Figure 2E, † need to corrected. Importantly, authors should be very careful to indicate all the statistics in data and legend (e.g. statistics missed in Figure 2E for MHPG and MHPG/NA groups).
Response: Symbols for the statistical test results were changed to be consistent throughout the graphs. Complete information for the statistical tests was included in the figure legends.
8. Figure 2B: further experiments are necessary for female mice.
Response: An additional experiment was done for female mice. Total n became eight for each genotype.
Reviewer #4 (Remarks to the Author): Summary: This manuscript reports an array of neurodevelopmental differences in mice lacking SLITRK1, which is a transmembrane protein associated with OCD-related disorders and known to function in neurite outgrowth and synapse formation. The paper builds on the authors' previously published work, which implicated noradrenergic mechanisms in the anxiety-like behaviors of slitrk1-deficient mice. Here, the authors report structural, behavioral, and neurochemical differences in slitrk1-deficient mice, with an emphasis on the neonatal prefrontal cortex. The authors also report two novel missense SLITRK1 mutations associated with schizophrenia and bipolar disorder and link these mutations to structural and functional deficits in the noradrenergic system. The paper is ambitious in scope, technically rigorous, and provides further evidence that cellular and molecular differences in noradrenergic signaling may contribute to numerous neurodevelopmental disorders, including OCD, schizophrenia, and bipolar disorder.

Major points:
This work is of interest to at least three specialized audiences -those who examine SLITRK protein functions in neurodevelopment, those interested in the development of the prefrontal cortex, and those interested in the cellular and molecular changes that contribute to OCD and other pervasive but poorly understood neurodevelopmental disorders. Two general issues regarding the clarity of the manuscript should be addressed. First, the final paragraph of the introduction should be revised and expanded to articulate the overall rationale and major findings of the paper with greater specificity. Second, the Results section could benefit from clear statements of rationale as each experiment is introduced and described. This was done nicely in the Discussion, but bears repeating in the Results as well.
Response: We revised the final paragraph as follows: "In this study, we firstly examined the neonatal phenotypes of Slitrk1-deficient mice. Because abnormalities in NA fiber development were observed, we investigated the molecular mechanism underlying Slitrk1-mediated control of the neurite development. Further, we conducted a re-sequencing analysis of patients with schizophrenia (SCZ) and bipolar disorder (BPD) to identify functionally defective and significantly enriched missense mutations. The analysis identified a SLITRK1 mutation that affects the NA fiber development-controlling and L1 family protein-binding abilities of SLITRK1. Finally, we sought to discuss the pathogenesis of OCRD, focusing on the role of neonatal NA-mediated neural circuit modification." We have added text that clarifies the logical flow in several subsections of the Result section.
In Figures 2 and 3   The claim that Slitrk1-knockout mice exhibit reduced GABAergic synapse density in the PFC is not adequately supported by the evidence presented in Figure S8. One or more additional inhibitory synapse markers would strengthen this claim considerably.
Response: We removed all results concerning the quantitative analysis of the synapse markers in Slitrk1 KO mice. Please see our first response.
Minor Points: In Figures 2-4, the WK labels below the X axes are redundant with the shading of the bars and the associated key. Removing the WK labels would improve the clarity of these figures.
Response: We removed the WK label, and the results in all the graphs are indicated in a consistent color code (WT = white, KO = red).